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ResearCh Methods in NeuroChemistry volumes A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new volume immediately upon publication. Volumes are billed only upon actual shipment. For further information please contact the publisher. Research Methods In Neurochemistry

EdiledbJ Neville Marks Center for Neurochemistry Rockland Research Institute Wards Island, New York, New York and Richard RDdnighl Department of Biochemistry Institute of Psychiatry University of London London, Great Britain VDlumea

PLENUM PRESS. NEW YORK·LONDON The Library of Congress cataloged the first volume of this title as follows:

Research methods in neurochemistry, v. 1- New York, Plenum Press, 1972- v. illus. 24 em.

1. Neu roc hem istry. QP356.3.R46 612'.8'042 72-222263

ISBN-13: 978-1-4615-7762-1 e-ISBN-13: 978-1-4615-7760-7 001: 10.1007/978-1-4615-7760-7

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher Contributors

GARY R. BARBARASH Section on Myelin and Brain Devel• opment, Developmental and Meta• bolic Neurology Branch, NINCDS, National Institutes of Health, Be• thesda, Maryland 20205

M. J. BRAMMER Department of Biochemistry, Institute of Psychiatry, De Crespigny Park, London, SE5 8AF, England

STEVEN W. BROSTOFF Department of Neurology, Medical University of South Carolina, Charleston, South Carolina 29425

MICHEL CHRETIEN Clinical Research Institute of Mon• treal, Montreal, Quebec H2W lR7 Canada

ROGER C. CAUSON Department of Clinical Pharmacol• ogy, Royal Postgraduate Medical School, University of London, Lon• don W12 OH5, England

CHARLES KENNEDY Laboratory of Cerebral Metabolism, National Institute of Mental Health, Public Health Service, Department of Health and Human Services, Be• thesda, Maryland 20205

v vi Contributors

RANDOLPH V. LEWIS Department of Biochemistry, Univer• sity of Wymoming, Laramie, Wyo• ming 82071

TRACY D. MAcINTOSH Section on Myelin and Brain Devel• opment, Developmental and Meta• bolic Neurology Branch, NINCDS, National Institutes of Health, Be• thesda, Maryland 20205

OLIVER E. PRATT Department of Neuropathology, In• stitute of Psychiatry, De Crespigny Park, London, SE5 8AF, England

RICHARD H. QUARLES Section on Myelin and Brain Devel• opment, Developmental and Meta• bolic Neurology Branch, NINCDS, National Institutes of Health, Be• thesda, Maryland 20205

VICTOR S. SAPIRSTEIN Biochemistry Department, Eunice Kennedy Shriver Center for Mental Retardation, Waltham, Massachu• setts 02254, and Department of Bio• logical Chemistry, Harvard Medical School, Boston, Massachusetts 02115

NABIL G. SEIDAH Clinical Research Institute of Mon• treal, Montreal, Quebec H2W 1R7 Canada

CAROLYN B. SMITH Laboratory of Cerebral Metabolism, National Institute of Mental Health, Public Health Service, Department of Health and Human Services, Be• thesda, Maryland 20205

LOUIS SOKOLOFF Laboratory of Cerebral Metabolism, National Institute of Mental Health, Public Health Service, Department of Health and Human Services, Be• thesda, Maryland 20205 Contributors vii

ALVIN S. STERN Department of Biopolymer Research, Roche Research Center, Hoffmann• La Roche Inc., Nutley, New Jersey 07110

MARY J. VOADEN Department of Visual Science, Insti• tute of Ophthalmology, University of London, London WC1H 9QS, Eng• land

MICHAEL J. WEISE Department of Neurology, Medical University of South Carolina, Charleston, South Carolina 29425 Preface

More than ever, the introduction of new methods or techniques serves to stimulate progress into understanding the structure and function of the nervous system. This axiom is exemplified by recent techniques that have revolutionized several branches of neurochemistry and promise to remain dominant for many years. Such developments underscore the need to remain abreast of new research strategies and provide further justification for the present series. The use of high performance liquid chromatography combined with bioassay methods provides a powerful technique for iso• lation and assay of trace amounts of neuropeptides. Two chapters in the present volume deal with this subject: one (Stenn and Lewis) describes the assay of enkephalins, and the other (Chretien and Seidah), on lipo• tropic peptides, includes procedures for structural analysis by microse• quencing. These methods rival earlier ones for peptide separations in speed, sensitivity, and cost and have general applicability in most labo• ratories. High performance liquid chromatography has also largely sup• planted earlier and more tedious procedures for the assay of catechola• mines, as described in Chapter 7 by Causon. As in earlier volumes, we have striven to retain a balance between studies on intact tissues and those on subcellular components. Studies on whole brain are represented by chapters on mapping procedures for glu• cose consumption utilizing deoxyglucose, are preeminently capable of correlating metabolic change with functional state (Sokoloff, Kennedy, and Smith), and on the use of continuous infusion methods to study the blood brain barrier system, a procedure that performs a useful supplement to those described in earlier volumes of the series using discontinuous methods (Pratt). Studies on isolated tissue retaining cellular organization are represented by the retina (Voaden), a useful chemical model that has attracted considerable attention in recent years and which can be used to correlate metabolic events with a sensory input. The latter studies may

ix x Preface be contrasted with those of Brammer for isolation of brain cells from frozen tissue and their use for biochemical measurements. With respect to specific components of the nervous system there are chapters by Sapirstein on carbonic anhydrase (a potential marker of glial cells), by Weise and Brostoff of isolation and characterization of peripheral myelin P2 basic protein and the domains responsible for induction of peripheral neuropathies, and by Quarles, Barbarash, and MacIntosh on methods for isolation and characterization of central or peripheral myelin. The last two chapters represent useful supplements to earlier contributions in the series on myelin and glycoproteins. As always it is a pleasure to record our deep gratitude to contributors for their perseverance and patience, and to Plenum Press for their en• couragement and cooperation.

Neville Marks, New York Richard Rodnight, London Contents

Section I ULTRASTRUCTURE AND FRAGMENTATION OF NEURAL TISSUES

Chapter 1 Techniques for Neurochemical Research on the Retina 3 Mary J. Voaden

I. The Retina as a Neurochemical Model ...... 3 II. Structure, Function, and Species Differences ...... 5 A. General Structure and Function ...... 5 B. Retinal Vascularization and the Blood-Retinal Barrier. . 8 C. Photoreceptor Cells and the Fovea ...... 9 D. Photoreceptor Outer Segment Turnover and Phagocytosis by the Pigment Epithelium...... 10 E. The Visual Pigments ...... 11 F. The Visual Cycle ...... 12 III. Functional Stimulation of the Retina...... 13 A. Photoreceptor Function: Stimulating Rods and Cones...... 13 B. Monitoring Retinal Function: The Electroretinogram .. 15 C. Working with a Dark-Adapted Retina ...... 18 IV. Studying the Retina in Vivo ...... 18 A. Intravitreal Injection ...... 19 B. Vitreal Perfusion ...... 20 C. Retinal Superfusion ...... 20 V. The Retina in Vitro ...... 21 A. Enucleation ...... 22

xi xii Contents

B. Eyes and Eye-Cups in Vitro ...... 22 C. Isolating the Retina ...... 22 D. Quantifying the Retina ...... 25 E. Maintenance of the Retina in Vitro ...... 26 F. Retinal Culture ...... 27 G. Techniques for Isolating and Studying the Retinal Pigment Epithelium ...... 28 VI. Fractionation of the Retina ...... 29 A. Retinal Fractionation by Cell Degeneration ...... 30 B. The Separation and Isolation of Retinal Cells ...... 34 C. Subcellular Fractionation of the Retina ...... 35 D. Tangential Sectioning of the Retina...... 36 VII. Rhodopsin ...... 38 A. Assaying Rhodopsin ...... 39 B. Optimizing Rhodopsin and Quantifying Opsin ...... 40 References ...... 41

Chapter 2 Isolation of Cells from Frozen Brain Tissue and Storage of Isolated Cells in the Frozen State ...... 49 M. J. Brammer

I. Introduction ...... 49 II. Events Occurring during Freezing of Cells: The Use of Cryopreservatives to Minimize Freezing Damage . . . . 50 III. Isolation of Cells from Frozen Brain Tissue and Freezing of Isolated Oligodendroglia ...... 52 A. Isolation of Oligodendroglial Perikarya from Whole Brain Stored at - 30 or - 80°C ...... 52 B. Isolation of Cells from Cryopreservative-Treated Gray and White Matter ...... 55 C. Freezing of Isolated Cells in the Presence of Cryopreservative ...... 57 IV. Properties of Isolated Cells: Assessment of Preservation of Cytoplasm ...... 58 A. Morphology of Isolated Cells ...... 59 B. Biochemistry of Isolated Cells ...... 67 V. Conclusions and Future Developments ...... 73 References ...... 74 Contents xiii

Section II PROPERTIES OF INTACT NEURAL TISSUES

Chapter 3 The Deoxyglucose Method for the Measurement of Local Glucose Utilization and the Metabolic Mapping of Functional Neural Pathways in the Central Nervous System ...... 79 Louis Sokoloff, Charles Kennedy, and Carolyn B. Smith

I. Introduction ...... 79 II. Theoretical Basis of Radioactive Deoxyglucose Method. " 80 III. Procedure ...... 84 A. Preparation of Animals ...... 87 B. Administration of [14C]Deoxyglucose and the Sampling of Arterial Blood ...... 88 C. Analysis of Arterial Plasma for [ 14C]Deoxyglucose and Glucose Concentrations ...... 88 D. Processing of Brain Tissue ...... 89 E. Preparation of Autoradiographs ...... 91 F. Densitometric Analysis of Autoradiographs ...... 93 G. Calculation of Rate of Glucose Utilization...... 93 IV. Theoretical and Practical Considerations ...... 94 A. Rate Constants ...... 94 B. Lumped Constant...... 97 C. Role of Glucose-6-phosphatase ...... 99 D. Influence of Varying Plasma Glucose Concentration· . . 100 E. Animal Behavior during the Experimental Period .... 101 V. Rates of Local Cerebral Glucose Utilization in the Normal Conscious State...... 102 VI. Effects of General Anesthesia ...... 102 VII. Relationship between Local Functional Activity and Energy Metabolism ...... 106 A. Increased Functional Activity-Experimental Focal Seizures...... 106 B. Decreased Functional Activity-Visual Occlusion . . .. 107 VIII. Computerized Color-Coded Image Processing...... 107 IX. The Use of the [14C]Deoxyglucose Method for Metabolic Mapping of Functional Neural Pathways ...... 109 X. Microscopic Resolution ...... 112 XI. ['8 F]Fluorodeoxyglucose Technique ...... 114 References ...... 114 xiv Contents

Chapter 4 Continuous-Injection Methods for the Measurement of Flux across the Blood-Brain Barrier: The Steady-State, Initial-Rate Method ...... 117 Oliver E. Pratt

I. Introduction ...... 117 II. Principle of the Method ...... 118 III. Development of a Procedure to Measure Flux across the Blood-Brain Barrier by the Steady-State, Initial-Rate Method...... 122 A. Background to the Problem...... 122 B. Improvements in Technique ...... 124 C. Derivation of an Injection Schedule to Maintain a Steady Level in the Circulation ...... 128 IV. Preliminary Preparation ...... 130 A. Preparation of the Animal ...... , 130 B. Preliminary Considerations ...... 131 C. Assessing Rate of Tracer Disappearance from the Bloodstream ...... 132 V. Devising a Suitable Injection Program ...... 133 VI. Implementation of the Injection Program ...... 135 A. An Apparatus Suitable for Giving Electronically Controlled Injections ...... 135 B. Checking the Effectiveness of the Injection Program. .. 137 C. Empirical Adjustment of the Infusion Program to Meet Altered Conditions ...... 137 VII. Measurement of Flux across the Blood-Brain Barrier. . .. 138 A. General Considerations ...... 138 B. Tissue Sampling and Tracer Assay ...... 139 VIII. Monitoring the Time Course of Tissue Tracer Uptake .. " 140 IX. Testing for Saturability of the Transport System ...... 141 X. Testing for Competitive Inhibition ...... 144 Xl. Discussion ...... 145 XII. Advantages ...... 146 XIII. Precautions ...... 148 References ...... " 149 Contents xv

Section III COMPONENTS OF NEURAL TISSUES-PEPTIDE HORMONES AND AMINES

Chapter 5 Methods for Isolation, Characterization, and Sequence Analysis of Enkephalin Precursors ...... 153 Alvin S. Stern and Randolph V. Lewis

I. Introduction ...... 153 II. Preliminary Purification Steps ...... 158 A. Chromaffin Granule Isolation ...... 160 B. Extraction Procedure ...... 162 C. Size-Exclusion Chromatography ...... ,...... 163 D. Assays ...... 163 III. Purification of Enkephalin-Containing Polypeptides: Reverse-Phase HPLC of Peptides and Proteins...... 168 A. Instrumentation ...... 168 B. High-Performance Liquid Chromatography Methods. .. 173 C. Application of Instrumentation and Methods...... 177 IV. Chemical Analysis of Enkephalin-Containing Polypeptides.. 181 A. Amino Acid Analysis ...... 183 B. Tryptic Mapping...... 184 c. Sequencing...... 185 V. mRNA-cDNA Cloning ...... 186 VI. Summary ...... 188 References ...... 189

Chapter 6 Microsequence of Polypeptide Hormones: Its Usefulness to Monitor the Isolation of Novel Molecules...... 195 Michel Chretien and Nabil G. Seidah

I. Introduction...... 195 II. Microsequencing ...... 196 A. General Comments ...... 196 B. Characterization from Pulse and Pulse-Chase Experiments ...... 196 xvi Contents

III. Application to Monitoring Purification of a New Pituitary Glycoprotein ...... 198 A. Methods...... 198 B. Results ...... 199 IV. Chemical Characterization ...... 203 A. Methods ...... 203 B. Results...... 204 V. Conclusion ...... 208 References ...... 208

Chapter 7 High-Performance Liquid Chromatographic Separation and Determination of Catecholamines ...... 211 Roger C. Causon

I. Introduction ...... 211 A. High-Performance Liquid Chromatography ...... 211 B. Analysis of Catecholamines ...... 213 II. Procedures ...... 214 A. Extraction and Concentration ...... 214 B. High-Performance Liquid Chromatography Systems. .. 216 C. Catecholamines Determined by HPLC with EC Detection ...... 227 D. Method for Human Plasma and Urine...... 229 III. Recent Developments ...... 230 IV. Conclusions ...... 236 References ...... 237

Section IV COMPONENTS OF NEURAL TISSUES-ENZYMES AND PROTEINS

Chapter 8 Purification of Brain Carbonic Anhydrase by Preparative and Immunologic Techniques ...... " 245 Victor S. Sapirstein

I. Introduction ...... 245 II. Enzyme Assay ...... 246 A. Solutions ...... 246 Contents xvii

B. Supplies ...... 247 C. Procedure ...... 247 III. Extraction of Soluble and Membrane-Bound Carbonic Anhydrase from Rat Brain ...... , 248 A. Solutions ...... 248 B. Supplies ...... 248 C. Procedure...... 248 IV. Preparation of Affinity Columns ...... 249 A. Solutions ...... 249 B. Supplies ...... 249 C. Procedure...... 249 V. Affinity Chromatography ...... 250 A. Solutions ...... 250 B. Supplies ...... 250 C. Procedure...... 250 VI. Analysis of Purified Material ...... 252 VII. Analytical Methods for the Isolation of Brain Carbonic Anhydrase ...... 253 A. Antibody Production ...... 253 B. Preparation of Immunoadsorbants ...... 257 C. Application of Immunoadsorbants ...... 257 VIII. Summary ...... 260 References ...... " 261

Chapter 9 Research Methods in Studies with the P2 Basic Protein ...... 263 Michael J. Weise and Steven W. Brostoff

I. Introduction ...... 263 II. Isolation and Characterization of P2 Protein ...... 264 A. Isolation and Purification Procedures ...... 264 B. Chemical Characterization of P2 ••••••••••••••••• 269 C. Characteristics of the :P2 Molecule ...... 275 III. Immunochemical Techniques in Studies of P2 Protein .... 281 A. Immunochemical Methods ...... 281 B. Localization of the Protein in Nervous System Tissue.. 287 IV. P2 in Studies of Experimental Allergic Neuritis ...... 292 A. Disease Induction Studies ...... " 292 B. Neuritogenic Domains of P2 Protein ...... 295 C. Immune Response to P2 •••••••••••••••••..•••• 296 D. Protection against EAN ...... 298 xviii Contents

V. Concluding Remarks ...... 299 References ...... 300

Chapter 10 Methods for the Identification and Characterization of Glycoproteins in Central and Peripheral Myelin ...... 303 Richard H. Quarles, Gary R. Barbarash, and Tracy D. MacIntosh

I. Introduction ...... " 303 II. Isolation of Myelin and Myelin-Related Fractions ...... 305 A. Isolation of Myelin ...... 305 B. Subfractions of Myelin and Myelin-Related Membranes ...... 306 III. Methods for Detecting Glycoproteins of Myelin ...... 311 A. Polyacrylamide Gel Electrophoresis ...... 311 B. Staining Glycoproteins with Periodic Acid-Schiff Reagents ...... 315 C. Labeling Glycoproteins in Myelin with Radioactive Precursors ...... 316 D. Binding of Radioactive Lectins to Myelin Glycoproteins on SDS Gels ...... 321 E. Tritium Labeling of Glycoproteins with Tritiated Borohydride ...... 324 IV. Distinguishing between Components That Are Genuine Components of Myelin Sheaths and Those That Are in Contaminants of the Isolated Myelin ...... 325 V. Purification and Characterization of Specific Glycoproteins ...... 329 A. Po Glycoprotein ...... 329 B. Myelin-Associated Glycoprotein ...... 331 C. Analytical Methods Used for Chemical Characterization ...... , 337 VI. Immunologic Procedures ...... 338 A. Preparation of Antibodies to MAG and Po ...... 338 B. Detection and Characterization of Antibodies and Antigents ...... 338 VII. Quantitation of Glycoproteins in Myelin ...... 346 A. Determination of Total Protein-Bound Carbohydrate in Myelin...... 346 Contents xix

B. Densitometric Measurement of Individual Glycoproteins on Polyacrylamide Gels ...... 347 C. Radioimmunoassay for the Myelin-Associated Glycoprotein ...... 350 VIII. General Comments and Conclusions ...... 354 References ...... 355

Index ...... 359